Growth of fungi on volatile aromatic hydrocarbons: environmental technology perspectives

The present study aimed the better understanding of the catabolism of monoaromatic hydrocarbons by fungi. This knowledge can be used to enhance the biodegradation of BTEX pollutants. Fungi with the capacity of using toluene as the sole source of carbon and energy were isolated by enriching environmental polluted soil and groundwater samples in solid state-like batches, air biofilters, and liquid cultures. Incubation conditions in the latter combined acidic pH and low water activity. The isolates were identified as Cladophialophora sp. (two strains), Exophiala sp., Leptodontium sp. and Pseudeurotium zonatum . The previously isolated toluene-growing fungus Cladosporium sphaerospermum was also included in the present study. The genera Cladophialophora , Exophiala (both anamorphs of Capronia ), and Cladosporium are classified into the black yeast-like and allied fungi. Members in this group are characterized for being cosmopolitan saprobes and opportunistic human pathogens. Growth curves showed that fungi grew on toluene with biomass doubling times of about 2 to 3 days. Some of the strains also grew on ethylbenzene and styrene. Kinetics of toluene degradation by cell suspensions followed the Michaelis-Menten model. Depending upon strain, the apparent KmSIZE=2>for toluene oxidation ranged from 5 to 22 μM. The toxicity of toluene, measured as the IC50 value, ranged from 2.4 to 4.7 mM. The metabolic pathway for the oxidation of toluene was studied in a variety of fungi by using fluorinated toluene analogSIZE=2>ues and by determining the formed metabolites by 19F NMR. Oxidation was similar in all toluene-assimilating fungi in which the initial conversion took place at the alkyl group. Toluene was initially oxidized to benzyl alcohol and benzoate, which was further hydroxylated at the ring first to 4-hydroxy-benzoate, and then to catecholic compounds. The latter served as substrates for the opening of the ring and further metabolism through the 3-oxoadipate pathway. The lack of significant benzene biodegradation by these fungi might be related with the incapacity of performing the ring hydroxylation. Oxidation of toluene at the aromatic ring was demonstrated for the zygomycete Cunninghamella echinulata . However, conversion rates here were very low in comparison with the assimilative metabolism and it occurred only co-metabolically.The substrates interactions during the degradation of BTEX mixtures were studied with the isolate Cladophialophora sp. strain T1. This fungus grew on a model water-soluble fraction of gasoline that contained all six BTEX components. Benzene was not metabolized but the alkylated benzenes (TEX) were degraded by a combination of assimilation and co-metabolism. Toluene and ethylbenzene were used as sources of carbon and energy whereas ortho - and meta -xylene were co-metabolized to phthalates as end-metabolites. Para- xylene was not degraded in complex BTEX mixtures but, in combination with toluene, it appeared to be mineralized. The metabolic profiles and the inhibitory nature of the substrate interactions indicated that TEX were degraded at the side-chain by the same monooxygenase enzyme. The growth of the strain T1 was also studied in sterile and non-sterile soil microcosms contaminated with a mixture of BTEX and MTBE. Inoculation with the fungus increased the degradation rates in soil after long exposure to BTEX and a low soil pH. Comparison of the biodegradation rates measured in presence and absence of indigenous bacteria, and the fungal inoculum suggested that the main interaction between indigenous and inoculated BTEX-degrading microorganisms was commensalistic. Alkylbenzenes were all degraded by the fungus, but benzene degradation required the activity of the indigenous soil microflora. MTBE could not be biodegraded. The presence and identity of the fungal inoculum in soil was confirmed at the end of the experiments by PCR-TGGE analysis of SSU of fungal 18S rDNA.Fungi utilizing aromatic hydrocarbons can advantageously be used for biodegradation of BTEX in air biofilters and in soil. Fungal biodegradation is similar in kinetic terms to that of bacteria and posses a higher tolerance to adverse environments. However, factors limiting the application of fungi concern the lack of benzene degradation and the potential pathogenicity to humans.